Composition and methods of esterified nitroxides gated with carboxylic acids

ABSTRACT

This invention is directed to the use of novel methods and nitroxide compositions having high redox activity to treat diseases and conditions related to free radicals. The compositions and methods described include pro-drug forms of nitroxide that enhance the therapeutic effect through their ability to penetrate readily into skin cells and remain sequestered within the cells. The formulations and methods described herein result in a significant accumulation of nitroxides in the skin cells, thus achieving a targeted delivery of a therapeutic or diagnostic dose. Applications using the novel compositions and methods described herein include use as improved imaging agents and as improved topical agents for treating conditions of the skin, including aging of the skin, wrinkles caused by sun-exposure, skin cancer, burns, psoriasis and alopecia, among others.

FIELD OF THE INVENTION

[0001] The present invention generally relates to compositions ofnitroxides and the therapeutic and diagnostic uses of speciallyformulated nitroxide compositions compatible with pharmaceutical use.These formulations are specifically designed for increased diagnosticand therapeutic utility when applied in a biological context whereintracellular retention is important and where hydrophobic barriers suchas skin, stratum corneum, or other such membranes are selectivelypermeable. These compositions are particularly useful when delivered toesterase-containing cells. These compositions may be used to alleviatethe toxic effects of free radicals in a living organism that result fromexposure to chemical agents and toxins, as well as sun, UV, or otherforms of ionizing radiation. Specialized methods and formulations alsoenable the ability to diagnose and treat a wide variety of physiologicalconditions, and to analyze in vivo reactions with imaging techniquesthat measure nitroxides and their reactivity. The invention also relatesto novel nitroxide compositions that permit targeting orcompartmentalization of a therapeutic dose of nitroxide within alocalized area, particularly penetration and retention within anintra-dermal interface in the skin.

BACKGROUND OF THE INVENTION

[0002] Radiation and free-radical mechanisms are the source for a widevariety of diseases and physiological damage to the body. Reactiveoxygen species such as the superoxide an ion (.O₂), the hydroxyl radical(.OH), hydrogen peroxide (H₂O₂), and singlet oxygen (¹O₂) are usuallyformed by ultraviolet or ionizing radiation and react through oxidationmechanisms to damage DNA, proteins, and biologically important lipids inthe body. The whole body effect of radiation and other such toxicreactive oxygen species is frequently manifested in damage to the skin.The acute effects include inflammation and the appearance of sun-damagedcells, principally as a result to overexposure to the sun. The exposureto reactive oxygen species also creates photosensitivity reactions andcan lead to immunological suppression when the overall exposure is largein quantity or duration. The long-term effects include photoaging,typically manifested through the appearance of the skin as well as atendency for certain physiological mechanisms that rejuvenate the skinand body to be compromised. Also, carcinogenesis is observed and theresulting skin cancers or keratoses can appear after years or evendecades following a significant exposure incident.

[0003] Skin may be viewed a major protective barrier that preferentiallyadmits some compounds and yet excludes external agents that may behazardous to a living organism, including toxic chemicals and freeradical species. The skin features certain endogenous protectivemechanisms against reactive oxygen species including the enzymessuperoxide dismutase, catalase, goutathione peroxidase, and reductase.Each of these enzymes acts to convert free radical oxygen species to acompound that is less toxic to the body and can ultimately bemetabolized through normal mechanisms. However, these enzymes are alsoinvolved in a cascade of free radical reactions that may inherentlyinvolve damage to the organism. Also, when an exposure to radiation orreactive oxygen species is particularly severe, these protectivemechanisms are often overwhelmed by the free radical cascade andsignificant cell or tissue damage or injury results.

[0004] Recognizing the potential damage from exposure to reactive oxygenspecies, several topical antioxidants have been developed that attemptto protect the skin from the majority of damage caused by reactiveoxygen species. Examples of the non-enzymatic antioxidants that may beapplied to the skin, and which exist in some quantities naturally,include vitamin C and vitamin E, para-amino benzoc acid (PABA) andothers. These topical antioxidants may reduce the acute effects ofexposure to radiation and may also, over time, reduce the chroniceffects observed with long-term or high dose exposure to radiation andreactive oxygen species.

[0005] The selective permeability creates unique problems and offersunique opportunities for drug administration. This environment of thedermal layers, as well as other membranes such as the enothelial layersof the vascular system and the blood/brain barriers, demonstrate acomplex drug clearance and pharmacodynamic and pharmacokineticparameters when exposure to ultraviolet and ionizing radiation causeschanges to normal cell metabolism.

[0006] The properties of an ideal topical antioxidant would includechemical stability, the ability to effectively penetrate both theepidermal and dermal layers of the skin, the maximum possible retentionin the target cells to maintain protective levels as long as possible,and, of course, overall lack of toxicity. The formulation of anantioxidant having all of these properties, that is also suitable fortopical administration, is difficult because free radical species aretypically extremely unstable and highly reactive. The high reactivity offree radical special is particularly problematic for therapeutic drugdesign because the physiological damage is caused by chemical reactionsbetween free radicals and body tissue that occur in very close proximityto the site where the free radical chemical species is generated. Theeffect of ultraviolet light upon molecular oxygen present in the dermallayers penetrated by UV light, for example, can produce high levels offree radicals in the skin that cause erythema and may ultimately lead toskin cancer and pre-mature skin aging (and wrinkling). Since molecularoxygen contains two unpaired electrons in its normal state, the effectof ultraviolet light upon triplet oxygen results in the creation of amore reactive oxygen species (i.e., singlet oxygen).

[0007] In addition to the direct damage to biological tissue componentssuch as structural proteins, superoxide radicals are extremely reactivewith a variety of molecules naturally present in the body, includingmembrane lipids and nucleic acids. Detrimental free radical reactionsresult in the alteration or loss of tissue and cell function, increasedpermeability of skin cell membranes, cell death, and cancer. See e.g.Canavese, C. et al., Int. J. Artif. Organs 10(6): 379-89 (1987). Manystudies have demonstrated that free radicals cause or aggravate a numberof other pathologic states, including cancer, ulcers, cataracts, closedhead injury, renal failure, injury to the nervous system,ischemia-reperfusion injury resulting from heart attack or stroke,shock, alopecia, sepsis, apoptosis, toxicity caused by certain drugsresulting from oxygen therapy in the treatment of pulmonary disease,psoriasis, the aging process, and many others. (See e.g. Dhalla, N. S.,Can. J. Cardiol. 15(5):587-593 (1999); Basaga, H. S., Biochem. CellBiol. 68(7-8): 989-98 (1990); Canavese, C. et. al., Int. J. Artif.Organs 10(6):379-89 (1987); Granger D. N. et. al., Gastroenterology81:22 (1981); Babior, B. M. et. al, J. Clin. Invest. 52:741 (1973)).

[0008] A particularly important chemical reaction in the pathophysiologyof skin damage occurs when nitric oxide and a superoxygen species reactto form peroxynitrite. Peroxynitrite was recently discovered topathologically activate epidermal growth factor receptor. Thesepathologies resulting in chronic lesioning of dermal tissue caneventually lead to DNA mutagenicity and cancer. For example, while itsetiology is unknown, free radicals have been implicated in the etiologyand/or symptoms of the chronic skin disorder, psoriasis. See e.g.Er-Raki A, et al., Skin Pharmacol. 6:253-258 (1993); Miyachi, Y. andNiwa, Y., Arch. Dematol. Res. 275:23-26 (1983). Psoriasis is accompaniedby increased levels of the enzyme xanthin oxidase in the epidermis ofthe skin, which itself is capable of generating oxygen free radicals.See Hersh, U.S. Pat. No. 6,011,067. Further, psoriasis patients displayan increase in the production of malondialdehyde, which is a reflectorof free radical damage in the body. Corrocher, Clin. Chim. ACTA 179:121(1989). Additionally, the effect of ultraviolet radiation from exposureto the sun on human skin is a growing concern since the majority ofchanges associated with an aged appearance result from chronicsun-damage. Warren et al., J. Am. Acad. Dermatol. 25:751-760 (1991);Frances, C. and Robert L., Int. J. Dermatol. 23:166-179 (1984).

[0009] Recently, topical retinoids became a popular treatment forsun-damaged skin, as well as for a wide variety of other skin disorders.See A. Haas et al., “Selected Therapeutic Applications of TopicalTretinoin”, JAAD, 15:870 (1986). Among the topical retinoids, Tretinoin(Retin-A or Renova®) is one of the most widely used, most commonly as ananti-wrinkle agent for sun-damaged skin. Tretinoin, however, has anumber of undesirable side effects including, ironically, heightenedsensitivity to sun exposure. See U.S. Pat. No. 5,721,275, Bazzano(1998). Further, even the lowest dose formulations of tretinoin can beunacceptable to certain individuals with sensitive skin since localirritation is a common side effect of tretinoin therapy. See U.S. Pat.No. 4,021,573 Lee (1977); 4,214,000 Papa (1980). Alpha-hydroxy acidshave also been used to treat various skin conditions, including sundamage. However, when used at high concentrations, the application ofthese formulations must be carefully monitored and can lead toinflammation, infection and scarring. See Morganti, P. et al., J.Applied Cosmetology 14(1):1-8 (1996); U.S. Pat. No. 4,247,547 Marks(1981). Even at lower concentrations, alpha hydroxy acids can cause skinsensitivity and irritation, and may require the user to use specialcosmetic and shampoo formulations due to this sensitivity. See e.g. U.S.Pat. No. 6,019,967 Breton et al. (2000).

[0010] However, clinical studies indicate that the ability of typicalagents to provide protection is variable. See Miyachi Y., J. Dermatol.Sci. 1995, 9:79-86; Bissett et al. Photodermatol. Photoimmunol.Photomed. 1990, 7:56-62. Moreover, the usefulness of many anti-oxidantsis limited by short duration of action in vivo, toxicity at effectivedosage levels, the inability of many compounds to cross cell membranes,and an inability to counter the effects of high levels of free radicals.For example, superoxide disuntates and catalase do not functioneffectively in the intracellular space, and procystein as a GSHprecursor, vitamins and other antioxidants are unable to alleviate theeffects of the high levels of free radicals encountered in injury anddisease and are rapidly bioreduced. Nitroxides are a class of stablefree radical compounds which have an unpaired electron maintained in anorbital configuration that yields both stability and unusual reactivity.Nitroxides exhibit antioxidant, enzyme-mimetic and catalytic activitiesin vitro and in vivo. (See Hsia U.S. Pat. Nos. 5,725,839; 5,741,893;5,767,089; 5,804,561; 5,807,831; 5,817,632; 5,824,781; 5,840,701;5,591,710; 5,789,376; 5,811,005; 6,048,967). The impact of smallmolecular weight nitroxides on a variety of reactive oxygen species(ROS)-induced diseases is also well documented in animal models.However, the intracellular and extracellular isotropical distributionand rapid bio-reduction of these nitroxides narrows their therapeuticindex and application. Further, where high dose, continuous infusionshave been employed, toxicity has been an issue that limits thetherapeutic utility. Small molecule nitroxides have been discovered toprotect cells against free radical-mediated damage, includinginflammation and injury caused by ionizing radiation, Mitchell U.S. Pat.No. 5,462,946 (1991); Hahn (1992), and post-ischemic reperfusion injury,Gelvan (1991). Certain low molecular weight nitroxides have also beenidentified that mimic the activity of superoxide dismutase (SOD), (A.Samuni et. al. J. Biol. Chem. 263:17921 (1988)) and catalase (R. J.Mehlhorn et. al., Free Rad. Res. Comm., 17:157 (1992)). Studies alsoshow that permeable nitroxides are capable of short-term protection ofmammalian cells against cytotoxicity from superoxide anion generated byhypoxanthine/xanthine oxidase and from hydrogen peroxide exposure. Seee.g. Noel-Hudson et al., Toxicol. Vitro. 3(2):103-109 (1989).

[0011] Previously, small molecular weight nitroxides have beencovalently attached to macromolecules, such as albumin, hemoglobin orstarch, at low molar ratios for non-therapeutic applications.Therapeutic efficacy can be achieved by covalently attaching thenitroxides at comparatively higher molar ratios (See Hsia U.S. Pat. Nos.5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831; 5,817,632;5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005; 6,048,967). Theresulting macromolecules have extended half-lives and demonstrate bothreduced toxicity and enhanced therapeutic effect in animal models ofvascular-related disease (e.g. stroke, gut ischemia) (21, 22) and haveutility in imaging and diagnostic applications. Application ofmacromolecular nitroxides, in combination with an isotropicallydistributes nitroxide (e.g. TPL), or its hydroxylamine derivative (e.g.TPA), results in a superior therapeutic index. This formulation exhibitsa spin-spin exchange between the macromolecular nitroxide and the cellpermeable nitroxide that occurs with a “halo” surrounding the vessel.Within the “halo” TPH reoxidizes to TPL. The creation of such a TPL“halo” and the corresponding increase in therapeutic index occursthrough the compartmentalization of cell-permeable and macromolecularnitroxides. However, unmodified membrane permeable nitroxides arerapidly reduced to an inactive form because a dynamic equilibriumbetween the nitroxide and its hydroxylamine state occurs in vivo, withthe equilibrium favoring the hydroxylamine state.

[0012] Thus, a major disadvantage of unbound small molecular weightnitroxides is their rapid reduction in vivo to a less activehydroxylamine derivative. Further, timely delivery of therapeuticnitroxide agents to cells in the affected region where oxygen freeradicals are rampant is required in order to produce a significanttherapeutic benefit. Small molecule nitroxides suffer from thedisadvantages mentioned above, principally a relatively short half-lifein vivo, difficulty in formulating such compounds to be selectivelycompartmentalized within the skin, and the tendency of such compounds tobe rapidly cleared from the dermal layers by bioreduction or clearancethrough the movement of fluids in the vascular interstitial tissuespace. See, “In vivo EPR Imaging of a Distribution and Metabolism ofNitroxide Radicals in Human Skin,” He et al., J. of Magnetic Resonance148, 155-164 (2001).

[0013] Due to the above problems, a need exists for improved nitroxidecompositions having high redox activity, tissue selectivity andcompartmentalization, favorable imaging characteristics, and lowtoxicity. A need exists for nitroxide compositions and methods whichdetoxify free radicals and related toxic species and which aresufficiently active and persistent in the body to avoid being rapidlyconsumed through bioreduction, clearance, or when increases in freeradical concentrations are encountered. Specifically, there is a needfor biologically compatible nitroxide compositions having high redoxactivity which can be sequestered in the skin so that a highertherapeutic or diagnostically effective concentration of nitroxide canbe attained and maintained.

SUMMARY OF THE INVENTION

[0014] This invention discloses the use of novel compositions comprisingstable esters of carboxylic acid linked nitroxides, or low molecularweight nitroxides, including derivatized molecules and precursors andderivatives thereof. The oxidation/reduction activity andcompartmentalization of stable nitroxides can be regulated by linking anegatively charged anion, such as a mono- or dicarboxylic acid group toa stable nitroxide molecule. The linkage of the negatively charged ionyields a “gate” function such that the permeability of the species ofcrossed cell membranes is altered and the in vivo half life is increasedsubstantially. In a preferred embodiment, the present invention exploitsthe use of carboxylic acid ester(s) of nitroxides for both sustainedbiological activity and targeted compartmentalization. The compounds canbe used advantageously in topical applications as a pro-drug fortargeting the intracellular compartment of esterase-containing cells. Inthe pro-drug form, the nitroxide compounds are administered, and afterpenetrating hydrophobic barriers (e.g. stratum corneum, brain bloodbarrier), the compounds are retained in target tissue cells due toreduced cell membrane permeability after hydrolysis of the ester groupsby cellular esterases. These compositions may be used to alleviateoxidative stress and to avoid the biological damage associated with freeradical toxicity including the effect of reactive oxygen speciesproduced by ultraviolet and ionizing radiation, exposure to toxins orchemicals or other free radical generators. These compounds reduceinflammation, apoptosis, and other cellular or tissue damage, both acuteand chronic. Due to their enhanced antioxidant and radioprotectantactivity, the compositions disclosed herein have increased therapeuticvalue compared to other topical antioxidants, which, in combination withtheir diagnostic value, allows the novel compositions and methods ofthis invention to be used advantageously in a wide variety ofapplications. These compounds can be used alone or in combination withother nitroxide containing species in therapy and diagnosis.

[0015] The nitroxide compositions of the present invention areformulated to be selectively membrane permeable and to be administeredas a precursor or derivative of the form of the nitroxide that persistsinside the cell. Upon administration, the precursor or derivative isconverted in vivo by intracellular enzymes to a therapeutically ordiagnostically active form, preferably a form that exhibits reducedmembrane permeability compared to the precursor, thereby maximizing thein vivo half-life and utility. Thus, the capability to maintain theconcentration of an active nitroxide in vivo pursuant to this inventionoffers advantages in virtually any application where administration of anitroxide is beneficial but the utility is limited due to rapidbio-reduction.

[0016] The drawbacks of conventional nitroxide formulations are overcomeby selecting a nitroxide having high redox activity and converting thenitroxide into an esterified derivative. In particular, this inventiondiscloses stable small molecule, or ion molecular weight nitroxideshaving unusually high redox activity and carboxylic acid and esterderivatives of these nitroxides formulated to be physiologicallycompatible and stable and non-toxic for pharmaceutical use. Whenformulated pursuant to the invention, the duration of diagnostic imagingpotential, as well as the therapeutic action, of nitroxides in the bodyis extended compared to unmodified nitroxides. In another embodiment, anitroxide ester, such as an ester of2,2,6,6,-tetramethyl-1-oxyl-piperdinene-4-succinate (“TOPS”) is appliedtopically while a macromolecular polynitroxide, such as polynitroxylatedalbumin, is also injected subcutaneously, intraperitoneally, orintravascularly. The polynitroxide albumin serves as an acceptornitroxide, and distributes predominantly in the vascular space and actsas a storehouse of activity. See Hsia U.S. Pat. No. 5,840,701, hereinincorporated by reference in its entirety.

[0017] The novel compositions described herein are also useful indiagnostic applications because the unpaired electron of a stablenitroxide is detectable by electron paramagnetic resonance spectroscopyand nuclear magnetic resonance spectroscopy. Imaging instrumentationcapable of detecting these compounds yield high quality images ofbiological structures. Pursuant to this invention, compositioncontaining nitroxide esters, polynitroxide macromolecules, and/or theirpro-drug analogs or derivatives yield active nitroxide levels in thebody that may be maintained for a prolonged period of time allowing bothimproved image contrast and longer signal persistence than withconventional nitroxide formulations. Furthermore, the selectivemodification of the chemical composition of the nitroxide molecule toallow predetermined compartmentalization and metabolism increases thediagnostic value of the nitroxide species.

[0018] Nitroxides having the structural compositions defined hereincontain chemical leaving groups that are metabolized in biologicalsystems to yield a modified nitroxide compound having different cellpermeability than the unmodified compound. Thus, the invention may alsobe characterized by the difference between two nitroxide species. Thefirst species contains a chemical group that is capable of beingmodified by intracellular biological processes such that a secondnitroxide species is produced in vivo. Typically, the first nitroxidespecies will be characterized by increased cell membrane permeabilityrelative to the second nitroxide species and the operative chemicalmodification takes advantage of this difference and the ability for thechemical modification to be susceptible to intracellular processes.Compounds of the invention include succinate, aspartate, andglutimate-linked forms of the nitroxide as well as other medium-sizedfatty acids capable of forming modified esters. The compounds may alsobe synthesized as mono or diesters and homo or hetero-esters. Forexample, experimental results presented herein show that topicalapplications of an ethyl ester or di-ethyl ester of2,2,6,6,-tetramethyl-1-oxyl-piperdinenel-4-succinate yields a targeted,diagnostic or therapeutic dose of nitroxide. The di-ethyl form of theester is a particularly useful form of the nitroxide for intracellularlocalization because the ester groups functions as leaving groups whenexposed to enzymes within cells. Pharmacokinetic results are provided toshow human skin penetration, compartmentalization, and retention ofDE-TOPS, increased plasma half-life ion toxicity, spectroscopicactivity, and a protective effect against reactive oxygen species.Topical application of DE-TOPS is also shown Animal data shows skinthickening, reduced apoptosis, and reduced skin wrinkling in acute andchronic radiation exposure models.

DESCRIPTION OF THE FIGURES

[0019] FIGS. 1A-1C show the structures of selected examples of speciallyformulated nitroxide compounds of the invention.

[0020]FIG. 1A is 2,2,6,6-tetramethyl-1-oxyl-piperidinenel-4-succinate(TOPS). FIGS. 1B and 1C are mono- (E-TOPS) and di-ethyl esters (DE-TOPS)of carboxylic acid derivatives.

[0021]FIG. 2 shows a liquid chromatograph spectrum identifying TOPS andits ester(s) of carboxylic acid derivatives, E-TOPS and DE-TOPS.

[0022]FIG. 3 shows the hydrolysis of DE-TOPS to TOPS in vitro sodiumhydroxide (NaOH) in an aqueous environment showing the promotion ofintracellular retention. As is apparent from the EPR signals, pre- andpost-hydrolysis, near complete conversion from DE-TOPS to TOPS occurs.

[0023]FIG. 4 compares the plasma half-life of a mono-ethyl esterifiedcarboxylic acid derivative of ¹⁴N-E-TOPS to ¹⁵N-TEMPOL.

[0024]FIG. 5 shows the extended in vivo plasma half-life of ¹⁴N E-TOPScompared to ¹⁵N TEMPOL when both are co-administrated intraperitoneally.

[0025]FIG. 6 shows the extended in vivo plasma half-life of E-TOPScompared to TEMPOL when both are co-administrated intravenously.

[0026]FIG. 7 shows the extended in vivo plasma half-life of E-TOPScompared to TEMPOL when both are co-administrated intramuscularly.

[0027]FIG. 8 shows the extended in vivo plasma half-life of E-TOPScompared to TEMPOL when both are co-administrated orally.

[0028] FIGS. 9A-C shows skin penetration and compartmentalization ofDE-TOPS in a Franz Diffusion cell when DE-TOPS and TEMPOL are co-appliedon hairless mouse skin: 9A—before application; 9B—in receptor cell; and9C in skin.

[0029]FIG. 10 shows human skin penetration and compartmentalization ofDE-TOPS.

[0030]FIG. 11 shows the relationship between the survival rate anddosage for E-TOPS compared to TEMPOL in an LD₅₀ study in mice.

[0031]FIG. 12 shows the metabolism of DE-TOPS through excretion in urineand reduction in plasma after topical application at 6 hours.

[0032]FIG. 13 shows the superoxide dismultase activity of E-TOPSdetermined by inhibition of the reduction of Cytochrome c.

[0033]FIG. 14 show that E-TOPS dose dependent inhibition ofhemoglobin-induced toxicity of cortical neurons.

[0034]FIG. 15 shows TOPS, E-TOPS, and DE-TOPS dose dependent inhibitionof peroxynitrite-induced toxicity on cortical neurons. Peroxynitritegenerated from 3-morpholinosydnonimine (SIN-1). Rat cortical neuronswere exposed to SIN-1 (1 mM) for 24 hours with or without the nitroxidesTOPS, E-TOPS, or DE-TOPS at 50 or 500 μM. Following exposure to SIN-1,cell viability was determined based on the cell-dependent formation ofpurple formazan from MTT with a one-hour incubation. Data aremeans±standard deviation for 12 determinations.

[0035]FIG. 16 shows that the nitration of hydroxy phenol acetic acid(HPA) by peroxynitrite was inhibited by TOPS, E-TOPS, and DE-TOPS in adose-dependent manner.

[0036]FIG. 17 shows that E-TOPS reduces apoptosis induced by tumornecrosis factor alpha (TNF-α) on Y-79 cell line at different incubationtimes.

[0037]FIG. 18 is a histopathological sample of mouse skin after 10 daysof chronic UVB radiation showing the inhibition of UVB-induced apoptosiswith topical application of DE-TOPS.

[0038]FIG. 19 shows that topical application of DE-TOPS prevents skinthickening induced by chronic UVB radiation compared with aplacebo-treated mouse. No significant difference appears between normaland DE-TOPS treated ice following 11 days of ultraviolet exposure.

[0039]FIG. 20 shows that the topical application of DE-TOPS accelerateswound healing compared with placebo treated mouse.

[0040]FIG. 21 shows that topical application of DE-TOPS prevents UVBinduced skin damage on mice.

[0041]FIG. 22 shows a comparison of DE-TOPS, a placebo, and Retin A 15days after treatment with DE-TOPS and ultraviolet radiation.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The term “nitroxide” is used herein to describe molecules havingstable nitroxide free radicals including precursors (such as the N-Hform), and derivatives thereof including their correspondinghydroxylamine derivative (N-OH) where the oxygen atoms are replaced witha hydroxyl group and exist in a hydrogen halide form. Stability of theunpaired electron is provided at the nitrogen nucleus by two adjacentcarbon atoms that are substituted with strong electron donor groups.With the partial negative charge on the oxygen of the N—O bond, the twoadjacent carbon atoms together localize the unpaired electron on thenitrogen nucleus. Nitroxides generally may have either a heterocyclic orlinear structure.

[0043] The physiological compartmentalization of the nitroxide pursuantto this invention can be achieved through several discrete chemicalstructures or molecular modifications. The molecules are designedpursuant to the criteria disclosed herein to provide the selectedpermeability and reactivity characteristics. Modified nitroxideformulations may be topically applied and targeted to specific cellsbecause the membrane solubility of the nitroxide is altered byconverting the nitroxide to the modified form as described herein. Oncethe modified nitroxide has entered the cell, the ordinary intracellularhydrolysis mechanisms of endogenous enzymes create derivatives of thenitroxide, which have reduced membrane permeability. Thus, thesecompounds readily enter the cell, but resist leaving the cell, and as aconsequence, exhibit increased permeability for transmembrane entry intoa target area and a decreased tendency to be removed from tissue byordinary physiological clearance processes. This feature yieldsselectable preferential compartmentalization in vivo and sustainedtherapeutic or diagnostic effect.

[0044] As described herein, accumulation and sequestration orcompartmentalization of nitroxide species may be enhanced by convertingthe nitroxide to carboxylic acid ester. This result can be optimizedusing negatively charged anions such as nono- or dicarboxylic acids.Topical applications prefer a diester, which may be asymmetric, andwhere one esterified group is more labile than the other. For example,the t-butyl ester nitroxide will be more readily hydrolyzed than ethylester because it is more labile than ethyl ester. Hydrolysis of anasymmetric nitroxide comprising these two esters will thus first yield aless membrane permeable mono-carboxyl TOPS. The selection of theparticular ester, e.g., methyl, ethyl, butyl, and mono or di-carboxylicacid derivative, e.g., succinate, aspartate, glutamate determines thecompartmentalization and intracellular accumulation characteristics andthus may be tailored to be higher or lower for specific diagnostic ortherapeutic indications. Likewise, preparation of nitroxide-diesters ofnaturally occurring dicarboxylic acids will permit increasedaccumulation and sequestration intracellularly and have an addedadvantage in that naturally occurring amino acids are well characterizedand bioreactivity is known for biodistribution, metabolism and excretion(ADME) studies.

[0045] The nitroxides that can be employed in this invention arestructurally diverse because the requisite property of the nitroxide isits ability to be chemically modified with carboxylic acids and viaesterification or to form the desired, modified nitroxide. Thus,nitroxides in their monocarboxylic, dicarboxylic, or polycarboxylicstate may be employed. Selected embodiments of the present inventionhave the following structures, although the invention contemplatesderivatives, isomers, substitutions, polymers, and other routinechemical modifications that preserve the functionality herein.

[0046] The carboxyl acid may be substituted with aspartate, glutimate,or another medium sized fatty acid and wherein R is a hydrogen moleculeor ester, a homo- or hetero- or mono- or diester, including the methyl-,ethyl-, propyl, isopropyl, butyl, and t-butyl forms, or other functionalequivalent.

[0047] Since the compounds described herein provide a targetedtherapeutic dose of antioxidant nitroxide, they are highly effective inpreventing and alleviating the effects of oxidative stress. E-TOPS wasfound to have an increased in vivo half life compared to TEMPOL viaintravenous administration, intramuscular, oral, and intraperitonealadministration at 200 mg/kg. See FIGS. 4, 5, 6, 7, and 8. The E-TOPS hasalso been demonstrated to have a very low acute toxicity profilecompared to TEMPOL in an LD₅₀ model in mice. See FIG. 11. In transdermalapplications, the DE-TOPS formulation reduces ultraviolet light inducedapoptosis, and ultraviolet light induced skin thickening in mice. Theseformulations also compare favorably to Retin A. See FIGS. 18-22. Asdescribed in more detail below, a major advantage of the nitroxidecompounds of the present invention is the ability to administer aphysiologically compatible solution containing these nitroxides in avariety of routes. Also, depending on the target cells and thediagnostic or therapeutic goal, the activity of these compounds may beenhanced by concurrent subcutaneous administration of a polynitroxide.

[0048] Compositions of the invention may be used indermatologically/cosmetically acceptable vehicles for transdermal suchas ointments, lotions, or gels, and other solvents or carriers acting asa dilutant, dispersant or carrier. The vehicle may comprise materialscommonly employed in skin care products such as water, liquid or solidemollients, silicone oils, emulsifiers, solvents, humectants,thickeners, powders, propellants and the like.

[0049] As noted above, the unpaired nitroxyl electron gives nitroxidesother useful properties in addition to the antioxidant activity. Inparticular, nitroxides in their free radical form are paramagneticprobes whose EPR signal can reflect metabolic information in biologicalsystems, e.g., oxygen tension or tissue redox states. Because naturallyoccurring unpaired electrons are essentially absent in vivo, EPR imaginghas the further advantage that there is essentially no background noise.Nitroxides also decrease the relaxation times of hydrogen nuclei, andare useful as contrast agents in proton or nuclear magnetic resonanceimaging (MRI). Nitroxides can also act as contrast agents to addmetabolic information to the morphological data already available fromMRI. For example, by substituting various functional groups on thenitroxide, it is possible to manipulate properties including relaxivity,solubility, biodistribution, in vivo stability and tolerance. Contrastenhancement obtained from nitroxide can improve the performance of MRIby differentiating isointense, but histologically dissimilar, tissuesand by facilitating localization and characterization of lesions, suchas blood brain barrier damage, abscesses and tumors.

[0050] In view of the stable chemical nature of the nitroxides, thecompositions disclosed herein can be administered to a patient byvarious routes. For the purposes of this invention, “pharmaceutical” or“pharmaceutically acceptable” compositions are formulated by knowntechniques to be non-toxic and, when desired, used with carriers oradditives that are approved for administration to humans. As describedin the Examples below, the routes of administration include intravenous,occular or intraoccular, oral, intramuscular, topical or transdermal,intraperitoneal or sub-cutaneous application or injection. Suchcompositions may include buffers, salts, or other solvents known tothese skilled in the art to preserve the activity of the vaccine insolution.

EXAMPLE 1

[0051] Synthesis and Preparation of Mono or Dicarboxylic Acid AndEsterified Nitroxide Species

[0052] The following chemical synthesis protocols yield stable nitroxidefree radicals whose physiological compartmentalization, as a function ofmembrane permeability and clearance in vivo, is regulated by anegatively charged anion such as mono- or di-carboxylic acids. Topicalapplications are particularly advantageous with ester derivatives thatprovide differential permeability across hydrophobic barriers with afirst nitroxide species having increased membrane permeability relativeto a second species having increased intracellular retention andantioxidant therapeutic utility after hydrolysis by intracellularesterases.

[0053] (a) Synthesis of Monoethyl andDiethyl2,2,6,6,-Teteramethyl-1-oxyl-4-piperidinyl succinate (E-TOPS) and(DE-TOPS).

[0054] A dry, two-necked flask fitted with a reflux condenser andmagnetic stirrer is charged with 45 ml of absolute tert-butanol and 6.72g of potassium tert-butoxide under nitrogen atmosphere. The mixture isboiled and heated under reflux until all solids are dissolved. The flaskis then cooled and 6.8 grams of 4-oxo-[TPO], 12 ml of diethyl succinateand 15 ml of tertiary butanol is added. The reaction mixture is thenheated for 10 minutes. After cooling with ice, and neutralizing withdilute HCL, the bulk of the alcohol is distilled off under reducedpressure. The residue is poured into 350 ml of ice water and acidifiedwith dilute hydrochloric acid to pH 3, and extracted with methylenechloride. The combined extracts are washed several times with a 1%ammonia solution. The solutions are cooled with ice, acidified andextracted again with methyl chloride. The resulting extract is thendried with sodium sulfate. After evaporation of the methylene chloride,the remaining oily red liquid is triturated with hexane. The crystal ofthe monoester is pressed on a porous porcelain plate and recrystallizedfrom a mixture of ether and hexane. The product is a yellow prism with amelting point of 103° C., and the expected yield is in the 60-70% range.

[0055] (b) Synthesis of t-butyl, ethyl2,2,6,6,-tetramethyl-1-oxyl-4-piperidinenel succinate (BE-TOPS).

[0056] DCC (dicyclohexylcarbodiimide, 1 equivalent) is added to asolution of E-TOPS and t-butanol (1 equivalent) in dry pyridine (50 ml).The reaction mixture is stirred for 4 hours and the precipitates areremoved by filtration. The filtrate is then evaporated to dryness andthe residue is purified by column chromatography.

[0057]FIG. 2 shows the separation of TOPS, E-TOPS, and DE-TOPS by liquidchromatography.

EXAMPLE 2

[0058] Hydrolysis of DE-TOPS to TOPS by Sodium Hydroxide (NaOH)

[0059] Referring to FIG. 3, the hydrolysis of DE-TOPS to thenon-esterified TOPS form will yield selective cell membrane permeabilityand increased intracellular retention when the nitroxide compounds areexposed to esterases or any intracellular enzyme or other biochemicalreaction that cleaves the ester group. The application of DE-TOPS as ahydrophobic pro-drug will penetrate the stratum corneum (dead cells)into the metabolically active base-membrane layers. Enzymatic hydrolysisof DE-TOPS will allow the product TOPS to be retained in the aqueousphase and hopefully and primarily in the intracellular volume. Todemonstrate this reaction, chemical hydrolysis of DE-TOPS is shown toyield a compound which preferentially distributes in water vs. octanol.

[0060] A 20 mg sample of DE-TOPS was added to a mixture of 1 ml waterand 1 ml octanol and allowed to partition. After 15 minutes, a 40 μlsample of water or oil was taken for EPR spectral analysis. Next, 20 mgof DE-TOPS was mixed in 1 ml of 10 mM NaOH and allowed to incubateovernight at room temperature. The solution was neutralized withhydrochloric acid and added to 1 ml of water and 2 ml of octanol. Themixture was allowed to partition, and after 15 minutes, a 40 μl sampleof water or oil was taken for EPR spectral analysis. EPR spectral weretaken using a Varian E9 spectrophotometer. Sweep width was 100 G,frequency was 9.535 GHz, microwave power was 2 mV and the modulationfrequency was 100 Hz.

[0061] Before hydrolysis, DE-TOPS was preferentially distributed inoctanol. The partition coefficient (LogP) for DE-TOPS was found to be1.7. After hydrolysis, the presumed product TOPS partitions in theaqueous phase. The partition coefficient (LogP) for TOPS was found to be−0.9. This shows that following hydrolysis, the product thus formed issignificantly hydrophilic and would be more readily compartmentalizedintracellularly.

EXAMPLE 3

[0062] In Vivo Plasma Half-Life of DE-TOPS and TEMPOL

[0063] As noted above, a principal drawback in existing nitroxide-basedcompositions for in vivo therapeutic or diagnostic use is the limitedhalf-life of these molecules and their rapid in vivo bioreduction andclearance. The result of the comparatively short half life of TEMPOL isa need to administer larger and larger doses to yield a profoundtherapeutic or diagnostic effect. As shown in FIG. 11 below, theincreased dosages of nitroxides can yield acute and chronic toxicity.However, where the plasma half-life of a compound is increased, theoverall dosage in both acute and chronic indications can be reduced.

[0064]FIG. 4 is an EPR spectra showing TEMPOL, and E-TOPS simultaneouslyrecorded at distinguished magnetic field positions. Plasma half-life ismeasured by collecting the spectrum each minute for 60 to 90 minutes.The peak height of ¹⁵N TEMPOL or ¹⁴N E-TOPS EPR signal is calculatedfrom each spectrum. The peak height of TEMPOL or E-TOPS is plottedagainst time as shown in FIGS. 5-8. Referring to FIG. 5, in anintraperitoneal administration, the effective therapeutic activity of¹⁴N E-TOPS (upper line) is substantially enhanced for the entirehalf-life of the compounds. Although ¹⁵N TEMPOL (lower line) has ameasurable half-life exceeding 40 minutes, E-TOPS has substantiallyhigher activity for at least 80 minutes. The doses are 125 mg/kg ofE-TOPS and 80 mg/kg TEMPOL at a 1:1 molar ratio.

[0065]FIG. 6 shows a measurement of the in vivo plasma half-life of anintravenous administration of E-TOPS and TEMPOL at 125 mg/kg. In theintravenous infusion example, TEMPOL (lower line) is rapidly reduced invivo such that, by the five minute mark after infusion, very littleactive TEMPOL remains in the intravascular space. By contrast, theactivity of E-TOPS (upper line) is measurably increased, compared toTEMPOL, for at least 50 minutes. Referring to FIG. 6, the extension ofthe in vivo plasma half-life of E-TOPS compared to TEMPOL is shown whenboth compounds are co-administered intravenously at a dose of 125 mg/kgof E-TOPS and 80 mg/kg of TEMPOL for a 1:1 molar ratio.

[0066] As is shown in FIG. 7, beyond the first few minutes the in vivoplasma half-life of E-TOPS is dramatically extended over TEMPOL for atleast 60 minutes following intramuscular co-administration.

[0067] Referring to FIG. 8, the in vivo plasma half-life of E-TOPScompared to TEMPOL is shown to be extended when both compounds areadministered orally. As in FIGS. 5-7, the doses are 125 mg/kg of E-TOPSand 80 mg/kg of TEMPOL at 1:1 molar ratio. As is indicated by FIG. 8,the in vivo plasma half-life of E-TOPS is dramatically extended comparedto TEMPOL following co-administration of the compounds and for at least40 minutes thereafter.

EXAMPLE 4

[0068] Penetration and Compartmentalization in Human Skin

[0069] Referring to FIG. 9, DE-TOPS (100 mM) in a petroleum base wasapplied on fresh human skin. The receptor buffer is PBS with 0.01%sodium azide. The buffer was constantly stirring. The cell wasmaintained at 37C with a circulating water bath. FIG. 9 shows the degreeof skin penetration. Mouse skin (or donor human skin) to cover the topof the receptor and cell DE-TOPS is applied on top of the skin. Underthe skin, PBS buffer is applied to keep the skin alive. 24 hours laterthe buffer will be collected for EPR assay. The surface of the skin iscleaned and the skin sample tested for EPR Signal. Although TEMPOL andDE-TOPS have same signal intensity prior to application, twenty-fourhours after application a stronger TEMPOL signal exists in the buffercompound to DE-TOPS. However, in the skin the E-TOPS signal is strongerthan TEMPOL. Thus, DE-TOPS is localized in skin compared with TEMPOL.DE-TOPS will penetrate into the cells of the epidermis and dermis whereit will be enzymatically hydrolyzed and become compartmentalized.Compared to the freely soluble Tempone, this would result in a moreuniform distribution of DETOPS in these skin layers.

[0070] Preliminary S-band EPR spectroscopy and imaging experiments usingDETOPS were performed on the skin of a human volunteer. The humanvolunteer's forearm skin, about 6 mm diameter circular spot, typicallyat the ulnar surface of the wrist, was washed thoroughly with alcoholand 3 μL of 100 mM DE-TOPS solution (about 2×10¹⁷ spins) was applied tothe marked skin area. Five minutes later, when the deposited solutiondried, a specially designed positioning holder with a 7 mm diameter welland bottom disk that locked into a well in the resonator cap wasattached to the skin to fix this region of skin to the surface resonatorSee, “In vivo EPR Imaging of a Distribution and Metabolism of NitroxideRadicals in Human Skin,” He et al., J. of Magnetic Resonance 148,155-164 (2001). EPR and EPRI measurements were then started.Measurements on the volunteer were performed for 15 to 20 minutesperiods after which there were 30-minute rest periods in which thevolunteer removed the arm from the resonator and the magnet. This holderfixed the skin positioning and assured a constant filling factor of theloaded resonator. The positioning holder was left attached to the armfor the entire series of measurements lasting up to 8 hours.

[0071] Referring to FIG. 10, a color-coded image of CNO penetration andcompartmentalization in human skin is shown. The 1-D spatial images wereobtained from the skin of the fore-arms of the same human volunteer at 1hr and 8 hr post-topical application of 3 μL or DE-TOPS (100 mM inDMSO). The measurements were performed using S-band (2.2 GHz) EPRimaging system with a specially designed surface resonator as reported(He et al., supra). The dotted line marks the surface of the skin. Theestimated skin depths are marked as epidermis, dermis and subcutaneouslayers. Compartmentalization results in a more diffuse distribution ofDE-TOPS throughout the skin layers. The 1-D EPR spatial image of DE-TOPScompared to tempone show an enhanced visual distribution throughout thedermis and epidermis by eight hours.

EXAMPLE 5

[0072] Acute Toxicity LD₅₀ of TEMPOL and E-TOPS.

[0073] As noted above, the practical, clinical application of unbound,small molecule nitroxides has been limited by the reduced activity invivo and comparatively short in vivo half-life. The result of thereduced in vivo half-life is the need to administer a larger dose toachieve the same therapeutic or diagnostic effect. The toxicity ofTEMPOL may be measured with an LD₅₀ model to determine the survival rateof mice at varying dosages. FIG. 11 shows the acute toxicity curve forTEMPOL as a function of survival rate with increasing dosages. TheE-TOPS formulation shows essentially no decrease in survival rate atdosages up to 3.5 mmol/kg whereas TEMPOL shows zero survival rate at thelower dosage of 2.0 mmol/kg. Significant differences in survival rateappear between the dosage of 2.0-2.5 mmol/kg. FIG. 12 shows the strongerEPR signal (proportional to concentration) from DE-TOPS in urinecompound for plasma after 6 hours, showing excretion and clearancethrough normal metabolism.

EXAMPLE 6

[0074] Inhibition of Cytochrome Reduction

[0075] E-TOPS is shown to be a hydrolytic intermediate of DE-TOPS and tofunction as a SOD-mimetic based on the inhibition of cytochromereduction by superoxide. The superoxide scavenging (SOD-mimetic)activity of E-TOPS was measured as the capacity to inhibit the reductionof cytochrome by superoxide radical. The reactions were analyzedcalorimetrically at 550 nm and 25° C. with an HP spectrophotometer. Thereactions were assayed with various concentrations of E-TOPS, 8×10⁻⁵ Mcytochrome, 1×10⁻⁴ M xanthine, 6 μg/ml catalase, and 28 mU/ml xanthineoxidase. All of the chemicals and enzymes were diluted into Hank'sBalanced Salt Solution (HBSS, Sigma). A reaction cocktail wasimmediately mixed by inversion in a cuvette and the absorption increaseat 550 nm was recorded over 5 minutes. A blank sample (withoutnitroxide) showed the maximum rate of absorption. The IC₅₀ wascalculated from the dose response curves as the concentration of boundor free nitroxide giving 50% inhibition of the maximum absorption rate.

[0076] Referring to FIG. 13, reduction of cytochrome by superoxideoccurs with increasing concentrations of E-TOPS. The correlationcoefficient by linear regression is r²=0.998 demonstrating that E-TOPShas SOD mimetic activity.

EXAMPLE 7

[0077] Hemoglobin Toxicity in Cultured Rat Cortical Neurons

[0078] E-TOPS is neuroprotective in a model of hemorrhagictransformation in stroke. Primary neuronal cultures were made fromforebrains of fetal rat pups (embryonic day 15). The cells weredispersed by repeated mechanical trituration in neuronal culture medium(MEM Eagle (Sigma, M4526), supplemented with glutamine (2 mM),penicilin-streptomycine (50 Units/ml -0.05 mg/ml), heat-inactivatedhorse serum (10%), fetal bovine serum (10%), glucose (0.5% or 28 mM).Following centrifugation (900 g; 5 min), the cells were placed ontopoly-L-lysine-coated 96 well plates at a density of 5×10⁶ cells/well.Hemoglobin in saline was added at 10 uM final concentration and ETOPSwere added at 10 uM, 1 uM, and 0.1 uM final concentration. 24 hoursafter incubation neuronal viability was quantitatively determined usingthe calorimetric MTT assay. MTT was added to each well such that thefinal concentration of the dye was 0.15 mg/ml. Plates were then returnedto the incubator for 1 hour at which time unincorporated MTT wasremoved, and the plates allowed to air dry. The purple formazan productpresent in viable cells was then dissolved by adding acidifiedisopropanol (with 0.1 N HCl in) and the absorbance intensity (540 nm)was measured using a 96 well plate reader. % Control=(Test A₅₄₀/MeanControl)×100%. FIG. 14 shows increased neuronal viability with theE-TOPS samples.

EXAMPLE 8

[0079] TOPS or TOPS-ester Neuroprotection of Cortical Neurons Exposed tothe Peroxynitrite Generator SIN-1.

[0080] SIN-1 toxicity is studied in cultured rat cortical neurons basedon the generation of peroxynitrite. The demonstration that TOPS orTOPS-ester are neuroprotective in this model, translates intonitroxide-dependent blockade of EGFR activation caused by SIN-1.

[0081] Primary neuronal cultures were made from forebrains of fetal ratpups (embryonic day 15). The cells were dispersed by repeated mechanicaltrituration in neuronal culture medium (MEM Eagle (Sigma, M4526),supplemented with glutamine (2 mM), penicilin-streptomycine (50Units/ml-0.05 mg/ml), heat-inactivated horse serum (10%), fetal bovineserum (10%), glucose (0.5% or 28 mM). Following centrifugation (900 g; 5min), the cells were placed onto poly-L-lysine-coated 96 well plates ata density of 5×10⁶ cells/well. Cytotoxicity was induced in the cellsaccording to a published procedure (Carroll et al 2000). SIN-1(3-morpholinosydnonimine, Sigma), a PN generator, was dissolved in 50 mMphosphate (pH 5.0) just prior to use, and added to each well to give thefinal concentration of 1 mM. TOPS, E-TOPS or DE-TOPS were added at theindicated concentration to each culture 15 minutes prior to SIN-1.Neuronal viability was quantitatively determined using the colorimetricMTT assay. MTT was added to each well such that the final concentrationof the dye was 0.15 mg/ml. Plates were then returned to the incubatorfor 1 hour at which time unincorporated MTT was removed, and the platesallowed to air dry. The purple formazan product present in viable cellswas then dissolved by adding acidified isopropanol (with 0.1 N HCl in)and the absorbance intensity (540 nm) was measured using a 96 well platereader. % Control=(Test A₅₄₀/Mean Control)×100%.

[0082] Referring to FIG. 15, TOPS or TOPS-esters prevented the toxicityof SIN-1 in a dose dependent manner. Neuroprotective concentrations ofthese compounds will prevent peroxynitrite-dependent EGFR activation bypreventing the covalent dimerization of receptors and their subsequentautophosphorylation.

EXAMPLE 9

[0083] Antioxidant Activity by Inhibition of Nitration

[0084] To demonstrate antioxidant activity of DE-TOPS, E-TOPS and TOPSin vitro, these compounds are compared with TEMPOL as the gold standard.TEMPOL was shown to prevent the nitration of 4-hydroxyphenylacetic acid(HPA) by peroxynitrite in vitro (Carroll et al., 2000). In thispreliminary experiment, % inhibition by TEMPOL, DE-TOPS, E-TOPS or TOPSof peroxynitrite-dependent nitration of HPA was measured.

[0085] Peroxynitrite was made by a procedure described previously (Reedet al., 1974). Solutions of 1 mM 4-hydroxyphenylacetic acid (HPA, Sigma)were made in 100 mM sodium phosphate at pH 6.5. Certain amount of TOPS,E-TOPS and DE-TOPS were added to 1 ml of HPA solution mentioned above togive a final concentration of nitroxide at 0.98, 3.91, 15.5, 62.5 and250M. Peroxynitrite was added at a final concentration of 1 mM to startthe nitration. Reactions were also carried out using inactiveperoxynitrite as blank and zero nitroxide as positive control. Thenitration was followed spectrophotometrically at 405 nm. Theconcentration of 4-hydroxy-3-nitrophenylacetate was determinedspectrophotometrically (430=4400 M⁻¹cm⁻¹) after the pH of reactionmixtures were increased to 10-11 with NaOH.

[0086] Referring to FIG. 16, 40% to 60% of HPA nitration by 1 mMperoxynitrite are inhibited by the nitroxides of the invention at 3 μM.The mechanism of inhibition by nitroxides is catalytic as was reportedby Carroll et al., 2000. As peroxynitrite is suggested to be animportant player in radiation-induced cellular damage, TOPS-esters hasutility as a therapy for skin exposed to reactive oxygen species such asperoxynitrite.

EXAMPLE 10

[0087] Cell Apoptosis Measured by Exposure to the TNF α

[0088] The UV light induced apoptosis may be measured by a model inwhich apoptosis is induced by exposure to tumor necrosis factor alpha(TNF-α). As shown in FIG. 17, measurement of apoptosis severity overtime for cells exposed to TNF-α plus E-TOPS is measured against acontrol. Cultured human Y-79 cells were maintained at pH 7.4 in cultureflasks in a mixture of amphotericin/penicillin/streptomycin treated (1%v/v) RPMI 1640 media with L-glutamine and 10% fetal bovine serum in anincubator under 10%-CO₂/90%-air brood conditions at 37 degrees Celsiusand 20% humidity. E-TOPS was prepared as a 10 mg/ml formulation. Fromthis stock solution, 10 μl was added to 1 ml of the cell suspensionsolution so that the final concentration of ETOPS was 100 μg/ml ofETOPS. Human TNF-α (10 g/1 ml) was used to prepare serial mediadilutions to obtain TNF-α concentrations of 5.0 ng/ml. Cell densitiesand viability were determined by trypan blue exclusion assay on a Zeissinverted microscope to ensure cell concentrations prior to cytometricassaying. After gentle mixing of the TNF-α, vial sets at the givenconcentrations for 6, 24, and 48 hours, the cytokine-treated cells wereresuspended in PBS. The cells were resuspended in annexin V-FITCconjugate solution and incubated at room temperature in the dark forfifteen minutes. Binding buffer was then added to each of the samples tobring the cell densities up to approximately 1.0×10⁶ cell/ml. Thebinding buffer was prepared by mixing the following together: 10 ml of1M HEPES/NaOH, ph 7.4, 30 ml of 5M NaCl, 5 ml of 1M KCl, 1 ml of 1MMgCl₂, 1.8 ml of 1M CaCl₂ and 52.2 ml of DDW.

[0089] Flow cytometric methods were employed to take advantage ofannexin V's reversible and calcium-dependent binding to negativelycharged phosphatidylserine (PS) residues in a 1:50 annexin to PS ratio.Assay at 488 nm on a Becton-Dickinson FACStar flow cytometer was thenconducted on the cells from the culture vials to determine the relativeproportions of cells that were nonviable. A two dimensional x-y contourplot was used to show populations of early apoptotic events separatefrom late apoptotic events. Compensation was set before the cytometrictrials by using an annexin-only stained population, a propidium-onlystained population, and an unstained population to delimit the ceilingsof detection in the respective FL-1/Fl-2 quadrants. Total samplingsaveraged 5000 cell events counted out of sample populations averagingwell over 1.0×10⁶ cells/ml with statistics and regression analysis givenfor each set of sample quadrants using CellQuest software. Fluoresceinstained populations alone demarcated apoptotic detection while dualcounterstaining with propidium iodide indicated necrotic populations.

[0090] Referring again to FIG. 17, FL1/FL2 plots demonstrate lightscattering showing that E-TOPS rescued (more cell survival) Y-79 cellsfrom increased apoptosis over time at 5.0 ng/ml TNF-α doses whencompared to control Forward and lacteral cytometric light scatteringcharacteristics also showed that E-TOPS exhibited protective effects inRb cell types. Negligible amounts of cells died due to necrosis asdefined by PI cytometric gating.

EXAMPLE 11

[0091] Uvb Induced Apoptosis

[0092] Measurement of the caspase 3 enzyme in SKH-1 mouse skin is aquantitative analysis of ultraviolet light induced apoptosis. SeeHurwitz et al., Experimental Dermatology 2000 June; 9(3):185-91. Topicalapplication was daily 10 minutes prior to UVB radiation. UVB dose was200 mJ/cm2 during 15 minutes. UVB lamp has spectrum distribution of290-31 0 nm at 80% below 310 nm at 20%. The DE-TOPS formulations are 100mM in a petroleum base. Referring to FIG. 18, the topical administrationof DE-TOPS inhibits UV induced apoptosis 10 days after chronic radiationwith ultraviolet B light.

EXAMPLE 12

[0093] Protection Against Ultraviolet Induced Skin Thickening andEnhanced Wound Healing

[0094] Referring to FIG. 19, 11 days exposure to UVB causes significantskin thickening compared to normal mouse skin without UVB exposure.Histopathological skin sections reveal that topical application ofDE-TOPS dramatically reduces skin thickening induced by UVB. Topicalapplication was daily 10 minutes prior to UVB radiation. UVB dose was200 mJ/cm2 during 15 minutes. UVB lamp has spectrum distribution of290-310 nm at 80% below 310 nm at 20%. The DE-TOPS formulations are 100mM in a petroleum base. Referring to FIG. 21, with a similar protocol,wound healing is enhanced.

EXAMPLE 13

[0095] Effectiveness of DE-TOPS for Alleviating UVB-Induced Skin Damage.

[0096] Hairless Rhino mice were subjected to a fifteen-day UVirradiation experiment in which test materials were applied to the skin.The nitroxide concentrations in this study were 100 mM in a petroleumbase applied topically and daily 10 minutes before UVB radiation. Themice were exposed to UVB irradiation of 200 mJ/cm2 for 15 minutes with aspectrum distribution of 80% 290-310 nm and 20% below 310 nm. Skinmorphology (extent of wrinkling) was examined daily and recorded byphotography prior to any daily treatment. Treatment of the dorsal skinwith DE-TOPS resulted in reduction of wrinkling and an improvedcutaneous histological appearance (see compare FIG. 21).

[0097] To determine the advantage of DE-tOPS compared to commerciallyavailable topical therapies, BE-TOPS, Retin-A, and a placebo wereadministered to the skin of mice exposed to alternate days of UVBradiation. Treatment with Retin-A resulted in a reduction in skinwrinkling compared with control animals (See FIG. 22). Although thephoto-sensitivity of Retin-A caused discoloration and flaking of thedorsal skin, cutaneous histological sections of the dorsal skin alsorevealed Retin-A treatment resulted in a reduction in the density ofopen and deep cysts.

[0098] BE-TOPS also has the advantage over Retin-A that DE-TOPS treatedskin is not UVB sensitive.

What is claimed is:
 1. A pharmaceutical composition comprising ananti-oxidative amount of an ester of2,2,6,6-tetramethyl-1-oxyl-piperdene-4-succinate in a pharmaceuticallycompatible composition.
 2. The composition of claim 1 wherein thepharmaceutically compatible composition is the form for topicaladministration selected from the group consisting of a gel, cream,ointment, and emulsion.
 3. The composition of claim 1 in apharmaceutically compatible vehicle for administration by a methodconsisting of oral, intramuscular, intraperitoneal, and intravenous. 4.The composition of claim 1 wherein the ester is an assymetric diester.5. The composition of claim 1 wherein the ester is an ethyl ester, adiethylester, or a tert-butyl ester.
 6. The composition of claim 1further comprising polynitroxide albumin.